PROJECT SUMMARY/ABSTRACT Cell-cell fusion is critical to the conception, development and physiology of multicellular organisms, and is involved in a variety of biological processes, such as fertilization, myogenesis, placenta development, bone remodeling, immune response, tumorigenesis, and aspects of stem cells-mediated tissue regeneration. Failure in cell fusion leads to defects such as infertility, congenital myopathy, osteopetrosis, immune deficiency, and pre-eclampsia. A mechanistic understanding of cell fusion is not only important for fundamental biology but may also provide basis for its manipulation in therapeutic settings. My lab has been using Drosophila myoblast fusion as a model to study the general mechanisms underlying cell fusion. We have made an unprecedented discover that cell fusion is an asymmetric process in which one cell (attacking cell) invades its fusion partner (receiving cell) using actin-propelled membrane protrusions to promote fusion pore formation. Building on insights we learned from myoblast fusion in vivo, we have reconstituted high-efficiency cell fusion in an otherwise non-fusogenic, non-muscle cell line and uncovered a novel function for invasive membrane protrusions in fusogen engagement. Furthermore, we have discovered dynamic mechanosensory responses in the receiving fusion partner and demonstrated that mechanical tension is a driving force for cell fusion. Our studies to date have provided significant insights into the function of the actin cytoskeleton in promoting cell membrane juxtaposition and fusion. In the next five years, we will expand our research into two new directions. First, we will extrapolate the mechanisms that we uncovered in Drosophila to mammals and investigate the potential function of the actin cytoskeleton in mammalian cell fusion, as well as how transmembrane fusogenic proteins coordinate with the actin cytoskeleton to promote cell fusion. Second, we will identify and characterize novel transmembrane proteins, including new fusogens, in cell fusion using the reconstituted cell-fusion culture system as a model. We will continue to use an interdisciplinary approach including genetics, molecular biology, biochemistry, biophysics, live imaging, super-resolution microscopy and electron microscopy in our proposed research. By expanding from Drosophila to mouse, and from the actin cytoskeleton to transmembrane proteins, our research will not only gain major new insights into the fundamental principles of cell-cell fusion, but also have far-reaching impact on a broad range of fields, including membrane biology, cell biology and developmental biology.